CN108165595A - A kind of preparation method of wheat embryo anti-oxidation peptide - Google Patents
A kind of preparation method of wheat embryo anti-oxidation peptide Download PDFInfo
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- 241000209140 Triticum Species 0.000 title claims abstract description 85
- 235000021307 Triticum Nutrition 0.000 title claims abstract description 85
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 65
- 210000001161 mammalian embryo Anatomy 0.000 title claims abstract description 42
- 230000003064 anti-oxidating effect Effects 0.000 title claims abstract description 26
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 38
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 38
- 239000000243 solution Substances 0.000 claims abstract description 30
- 238000000034 method Methods 0.000 claims abstract description 26
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 26
- 239000012141 concentrate Substances 0.000 claims abstract description 21
- 102000004190 Enzymes Human genes 0.000 claims abstract description 18
- 108090000790 Enzymes Proteins 0.000 claims abstract description 18
- 229940088598 enzyme Drugs 0.000 claims abstract description 18
- 102000057297 Pepsin A Human genes 0.000 claims abstract description 15
- 108090000284 Pepsin A Proteins 0.000 claims abstract description 15
- 229940111202 pepsin Drugs 0.000 claims abstract description 15
- 230000009849 deactivation Effects 0.000 claims abstract description 9
- 238000000605 extraction Methods 0.000 claims abstract description 8
- 238000000227 grinding Methods 0.000 claims abstract description 7
- 159000000011 group IA salts Chemical class 0.000 claims abstract description 5
- 239000012266 salt solution Substances 0.000 claims abstract description 5
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 4
- 239000007788 liquid Substances 0.000 claims description 13
- 238000004321 preservation Methods 0.000 claims description 11
- 239000012528 membrane Substances 0.000 claims description 10
- 239000000843 powder Substances 0.000 claims description 10
- 238000000926 separation method Methods 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 9
- 239000007787 solid Substances 0.000 claims description 8
- 238000005119 centrifugation Methods 0.000 claims description 7
- 238000001914 filtration Methods 0.000 claims description 6
- 230000008569 process Effects 0.000 claims description 6
- 239000012460 protein solution Substances 0.000 claims description 6
- 150000001447 alkali salts Chemical class 0.000 claims description 5
- 230000008859 change Effects 0.000 claims description 4
- 238000004255 ion exchange chromatography Methods 0.000 claims description 4
- 238000002386 leaching Methods 0.000 claims description 3
- 230000006835 compression Effects 0.000 claims description 2
- 238000007906 compression Methods 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 238000001641 gel filtration chromatography Methods 0.000 claims description 2
- 230000007062 hydrolysis Effects 0.000 claims description 2
- 238000006460 hydrolysis reaction Methods 0.000 claims description 2
- 239000011344 liquid material Substances 0.000 claims description 2
- 230000007935 neutral effect Effects 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 5
- 238000010266 Sephadex chromatography Methods 0.000 abstract 1
- 235000018102 proteins Nutrition 0.000 description 31
- 102000004196 processed proteins & peptides Human genes 0.000 description 21
- 229920001184 polypeptide Polymers 0.000 description 19
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 14
- 239000004365 Protease Substances 0.000 description 9
- 102000035195 Peptidases Human genes 0.000 description 7
- 108091005804 Peptidases Proteins 0.000 description 7
- 230000003078 antioxidant effect Effects 0.000 description 7
- 230000007760 free radical scavenging Effects 0.000 description 7
- 235000019419 proteases Nutrition 0.000 description 7
- 239000011780 sodium chloride Substances 0.000 description 7
- 229920005654 Sephadex Polymers 0.000 description 6
- 239000012507 Sephadex™ Substances 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 5
- 238000005227 gel permeation chromatography Methods 0.000 description 5
- 238000009777 vacuum freeze-drying Methods 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 4
- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 description 4
- 229920002271 DEAE-Sepharose Polymers 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 150000003254 radicals Chemical class 0.000 description 3
- 108090000526 Papain Proteins 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
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- 238000004458 analytical method Methods 0.000 description 2
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- 150000001875 compounds Chemical class 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 210000004051 gastric juice Anatomy 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 description 2
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- BQJCRHHNABKAKU-KBQPJGBKSA-N morphine Chemical compound O([C@H]1[C@H](C=C[C@H]23)O)C4=C5[C@@]12CCN(C)[C@@H]3CC5=CC=C4O BQJCRHHNABKAKU-KBQPJGBKSA-N 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 229940055729 papain Drugs 0.000 description 2
- 235000019834 papain Nutrition 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 230000002000 scavenging effect Effects 0.000 description 2
- 238000002137 ultrasound extraction Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108010059892 Cellulase Proteins 0.000 description 1
- 108010061711 Gliadin Proteins 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
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- 206010020772 Hypertension Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000002785 anti-thrombosis Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 108010050792 glutenin Proteins 0.000 description 1
- 239000008236 heating water Substances 0.000 description 1
- -1 hydroxyl radical free radical Chemical class 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 238000001696 ion exchange chromatographie Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 229960005181 morphine Drugs 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000010183 spectrum analysis Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/18—Ion-exchange chromatography
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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Abstract
The present invention discloses a kind of preparation method of wheat embryo anti-oxidation peptide, and wheat embryo using micro-wave vacuum and is carried out ultramicro grinding by this method, adds in alkaline salt solution and wheat plantule protein is extracted；Extracting solution and raffinate are detached after extraction, extracting solution is concentrated by ultrafiltration, concentrate is digested to obtain wheat embryo activity peptide solution using pepsin, after enzyme deactivation and secondary concentration, obtain wheat germ peptide concentrate, it is isolated and purified with sephadex chromatography, ion-exchanger chromatography, the wheat embryo anti-oxidation peptide purified after dry.The protein extracting ratio of wheat embryo is high in the method for the present invention, and wheat embryo anti-oxidation peptide has good activity and application value.
Description
Technical field
The present invention relates to food processing technology fields, specifically provide a kind of preparation method of wheat embryo anti-oxidation peptide.
Background technology
Wheat embryo is one of principal by product of wheat processing industry and the most part of wheat nutrition, accounts for wheat seed
The 2~3% of grain, rich in protein, carbohydrate, fat, ash content and the physiological activity such as amino acid and some vitamins into
Point, it is referred to as the essence of wheat and nutrition treasure-house.Wheat plantule protein content is high, up to 30% or so, soybean is only second to, containing clear
Albumen, gliadin and glutenin etc., water soluble protein accounts for more than 30%.It is most beneficial after the proteolysis of wheat embryo
Ingredient for biologically active peptide, it be to the vital movement of living organism beneficial to or with physiological action peptides, again
Claim Functional Polypeptides.Active peptide by 20 kinds of different aminoacids of human body with variety classes and different number arrangement form, along with may be used also
Can there are two level, tertiary structure, type is very huge, each active peptide all has unique composition structure, different activities peptide
Composition structures shape its different bioactive functions.The mechanism of absorption of biologically active peptide is better than amino acid, has amino
The incomparable bioactive functions of acid mainly include class morphine sample, hormone and adjust hormone, adjusting and inhibit enzyme, exempt from present
Epidemic disease adjusting, anti-hypertension, norcholesterol anticancer, anti-oxidant and removing free radical, improves element absorption and minerals fortune at antithrombotic
The biologically active peptides such as defeated, promotion growth.Wheat embryo is not fully utilized at present, has potential intensive processing value.
Invention content
In view of the deficiencies of the prior art, the present invention proposes a kind of preparation method of wheat embryo anti-oxidation peptide, and this method is adopted
With basic salt method ultrasound assisted extraction wheat plantule protein, protein extracting ratio height；The different enzyme of Integrated Selection is to wheat embryo egg
Digested in vain, preferably go out pepsin as enzyme source, the preparation of polypeptide is carried out under the conditions of human body hydrochloric acid in gastric juice is simulated, extraction with
Complex optimum various process parameters in enzymolysis improve the utilization rate of wheat embryo.Specific technical solution is as follows：
A kind of preparation method of wheat embryo anti-oxidation peptide, which is characterized in that this method includes the following steps：
(1) after wheat embryo being carried out micro-wave vacuum, ultramicro grinding, wheat embryo Ultramicro-powder is obtained；Using alkalinity
Salt method extracts wheat germ powder with reference to ultrasonic wave added method, obtains wheat plantule protein solution；
(2) separation of solid and liquid takes supernatant, as wheat plantule protein extracting solution；
(3) the wheat plantule protein extracting solution of step (2) is concentrated by ultrafiltration, obtains wheat plantule protein concentrate；
(4) pepsin is added in above-mentioned concentrate, carries out the enzymolysis of albumen, enzyme deactivation after enzymolysis obtains germ protein
Peptide solution；
(5) wheat plantule protein peptide solution is concentrated by ultrafiltration, chromatographs filtering and drying, the wheat embryo purified
Anti-oxidation peptide.
Further, the leaching process described in the step (1) is specially：Using alkaline salt solution and wheat germ powder
Liquid material volume ratio be 5~15：1, heating water bath, extraction time 30-120min, extracting times 2~3 times.
Further, the pH value range of alkaline salt solution is 8-11 used by the step (1) neutral and alkali salt method, salt
A concentration of 0.2-1.2mol/L.
Further, the ultrasonic power in the step (1) is 30W-5KW, ultrasonic time 30-120min.
Further, the step (2) carries out separation of solid and liquid using the method for centrifugation or plate compression.
Further, the step (3), (5) ultrafiltration concentration operating pressure for 0.1-0.7MPa, ultrafiltration membrane aperture
For 10-200nm.
Further, hydrolysis temperature is 30-60 DEG C, enzymolysis time 1-6h in the step (4), and enzyme adding proportion is step
Suddenly the 0.1-1% of (3) concentrate.
Further, the enzyme deactivation condition in the step (4) is 80-100 DEG C of heat preservation 5-20min.
Further, the chromatography filtering in the step (5), using gel filtration or ion exchange chromatography
The combination of filtering, or both.
Compared with prior art, beneficial effects of the present invention are as follows：
For the present invention using basic salt method ultrasound assisted extraction wheat plantule protein, protein extracting ratio is high, and Integrated Selection is not
Same protease, and Integrated comparative has been carried out to the effect of protease, screening obtains pepsin, under the conditions of human body hydrochloric acid in gastric juice is simulated
The preparation of anti-oxidation peptide is carried out, is extracting the technological parameter with complex optimum in enzymolysis, improves the utilization rate of wheat embryo, tool
There is good application value；And the method for system integration ultrafiltration concentration, gel permeation chromatography and ion-exchange chromatography has obtained mesh
Mark product anti-oxidation peptide solution.
Description of the drawings
Fig. 1 is the process flow chart of the present invention；
Fig. 2 is the enzymolysis Optimizing Process Parameters interaction diagram of embodiment 1.
Fig. 3 is the gel chromatography figure of the separating obtained anti-oxidation peptide of embodiment 1；
Fig. 4 is 2 ion-exchange chromatography figure of embodiment；
Fig. 5 is the molecular weight mass spectrogram of polypeptide after embodiment 2 detaches；
Fig. 6 is the molecular weight mass spectrogram of polypeptide after embodiment 4 detaches.
Specific embodiment
Below according to attached drawing and the preferred embodiment detailed description present invention, the objects and effects of the present invention will become brighter
In vain, below in conjunction with drawings and examples, the present invention will be described in further detail.It is it should be appreciated that described herein specific
Embodiment is only used to explain the present invention, is not intended to limit the present invention.
Embodiment 1
As shown in Figure 1, the preparation method of the wheat embryo anti-oxidation peptide of the present invention is specific as follows：
(1) after wheat embryo being carried out micro-wave vacuum, ultramicro grinding, wheat embryo Ultramicro-powder is obtained, took 60 mesh
The sample 50g of sieve, using 0.5mol/L sodium chloride, pH value 10, solid-liquid ratio 1：10, sample is extracted using ultrasonic wave added method
60min, ultrasonic wave use 40W, and extraction twice, merges leaching liquor, obtains wheat plantule protein solution；
(2) separation of solid and liquid is carried out using 10000rpm centrifugations 30min, takes supernatant, as wheat plantule protein extracting solution；
(3) the wheat plantule protein extracting solution 900mL that step (2) obtains is taken to be concentrated by ultrafiltration, 40 DEG C of temperature, operation pressure
Power is 0.1MPa, and ultrafiltration membrane aperture is 200nm, is concentrated into 260mL, obtains wheat plantule protein concentrate；
(4) 50mL wheat plantule protein concentrates are taken, the pepsin for accounting for that concentrate mass ratio is 1% is added in, adjusts pH
It is 1.1 to be worth, and temperature is 60 DEG C, heat preservation enzymolysis 73.8min, and the antioxidant activity of supernatant, and 100 DEG C of enzyme deactivations are measured after enzymolysis
5min obtains germ protein peptide solution；
(5) step (4) acquisition solution being concentrated by ultrafiltration, operating pressure 0.4MPa, ultrafiltration membrane aperture is 200nm,
And using the filtering of Sephadex G-75 gel chromatographies, the isolated polypeptide portion with antioxidant activity carries out infrared vacuum
Freeze-drying, the wheat embryo anti-oxidation peptide purified.
Comparative example 1
Specific steps are identical with embodiment, and the pepsin in step (4) is only changed into 1% papain, adjust
PH value 7, temperature are 60 DEG C, heat preservation enzymolysis 2h.
Comparative example 2
Specific steps are identical with embodiment, only change the pepsin in step (4) into 1% cellulase, adjust pH value
It is 4.5, temperature is 60 DEG C, heat preservation enzymolysis 2h.
Comparative example 3
Specific steps are identical with embodiment, only change the pepsin in step (4) into 1% alkali protease, adjust pH
It is 9 to be worth, and temperature is 60 DEG C, heat preservation enzymolysis 2h.
Comparative example 4
Specific steps are identical with embodiment, only change the pepsin in step (4) into 1% compound protease, adjust pH
It is 7.0 to be worth, temperature 60 C, heat preservation enzymolysis 2h.
Centrifugation measures the antioxidant activity of supernatant after enzymolysis, as a result as shown in table 1 below.From table 1 it follows that stomach egg
The clearance rate of free radical optimizes the usage amount and condition of pepsin, obtains far above other several enzymes after white enzyme enzymolysis
Optimized parameter into enzymolysis process, as shown in Figure 2, it can be seen that enzymolysis time, temperature and three factors of pH value are mutual
Reciprocation, it is beneficial to the selection for most preferably digesting parameter.Sample substep is subjected to Sephadex G-75 gel permeation chromatographies,
As shown in Figure 3, it can be seen that in each section sample after gel chromatography, absorbance difference shows the difference of peptide content, to receiving
The different samples of collection carry out antioxidant activity analysis, merge the high each group sample of antioxidant activity, carry out infrared vacuum refrigeration and do
It is dry, obtain polypeptide sample.
Influence of the different enzymes of table 1 to hydroxyl radical free radical clearance rate
| Enzyme class | Clearance rate (%) |
| Pepsin | 40.0 |
| Papain | 12.8 |
| Cellulose protease | 0 |
| Alkali protease | 9.88 |
| Compound protease | 13.75 |
Embodiment 2
(1) after wheat embryo being carried out microwave vacuum freeze drying, ultramicro grinding, wheat embryo Ultramicro-powder is obtained, was taken
The sample 50g of 60 mesh sieve, using 0.8mol/L sodium chloride, pH value 10, solid-liquid ratio 1：8, sample is carried out using ultrasonic wave added method
100min is extracted, ultrasonic wave uses 60W, repeats extraction 1 time, obtains wheat plantule protein solution；
(2) separation of solid and liquid is carried out using 10000rpm centrifugations 60min, takes supernatant, as wheat plantule protein extracting solution；
(3) the wheat plantule protein extracting solution 800mL that step (2) obtains is taken to be concentrated by ultrafiltration, 40 DEG C of temperature, operation pressure
Power is 0.7MPa, and ultrafiltration membrane aperture is 10nm, is concentrated into 300mL, obtains wheat plantule protein concentrate；
(4) 50mL wheat plantule protein concentrates are taken, the pepsin for accounting for that concentrate mass ratio is 0.15% is added in, adjusts
PH value is 1.0,60 DEG C of heat preservation enzymolysis 80min, is heated to 100 DEG C of boilings enzyme deactivation in 5 minutes, obtains germ protein peptide solution；
(5) step (4) acquisition solution being concentrated by ultrafiltration, operating pressure 0.7MPa, ultrafiltration membrane aperture is 10nm, and
It is chromatographed and filtered, and carry out infrared vacuum freeze drying using DEAE-Sepharose F.F ion-exchangers, obtain polypeptide sample.
It is eluted with 0.1-0.5mol/L sodium chloride, as shown in figure 4, figure 4, it is seen that DEAE-Sepharose F.F matrix
Polypeptide is efficiently separated, downward trend after first increasing is presented in the active peptide eluted as time went on, to wherein
Eluent near peak is collected and numbers (1-5), measures it and removes the ability of free radical, the results are shown in Table 2, table
Bright 5th part Scavenging ability is most strong, infrared vacuum freeze drying is carried out as target polypeptides sample, through laser solution
It is 450.3 to analyse the mass spectral analysis sample segment polypeptide molecular weight mass-to-charge ratio, as shown in Figure 5.
The different DPPH free radical scavenging activities for collecting part (number 1-5) of table 2
| Number | DPPH free radical scavenging activities | Hydroxyl radical free radical Scavenging activity |
| 1 | 8.85 | 1.59 |
| 2 | 9.85 | 9.88 |
| 3 | 16.4 | 11.1 |
| 4 | 22.2 | 11.6 |
| 5 | 34.7 | 14.3 |
Embodiment 3
(1) after wheat embryo being carried out micro-wave vacuum, ultramicro grinding, wheat embryo Ultramicro-powder is obtained, took 60 mesh
The sample 100g of sieve, using 0.2mol/L sodium chloride, pH value 8, solid-liquid ratio 1：5, sample is extracted using ultrasonic wave added method
120min, ultrasonic wave use 30W, repeat extraction 1 time, obtain wheat plantule protein solution；
(2) separation of solid and liquid is carried out using 10000rpm centrifugations 60min, takes supernatant, as wheat plantule protein extracting solution；
(3) the wheat plantule protein extracting solution 950mL that step (2) obtains is taken to be concentrated by ultrafiltration, 40 DEG C of temperature, operation pressure
Power is 0.1MPa, and ultrafiltration membrane aperture is 200nm, is concentrated into 500mL, obtains wheat plantule protein concentrate；
(4) pepsin of concentrate mass ratio 0.1% is accounted for the addition of wheat plantule protein concentrate, adjusting pH value is
1.0,30 DEG C of heat preservation enzymolysis 360min, 100 DEG C are boiled 5min enzyme deactivations, obtain germ protein peptide solution；
(5) solution that step (4) obtains is concentrated by ultrafiltration, operating pressure 0.1MPa, ultrafiltration membrane aperture is
200nm.Polypeptide concentrate 200mL is obtained, using sephadex Sephadex G-100 and Sepharose F.F ion exchanges
Chromatography, sephadex Sephadex G-100 use 0.1- with pure water elution, Sepharose F.F ion-exchange chromatographies
0.5mol/L sodium chloride is eluted, and obtains the polypeptide isolated and purified.Infrared vacuum freeze drying is carried out after collecting filtrate, is obtained
Antioxidation polypeptide sample.Sample its molecular weight results after MALDI-TOF-MS is tested and analyzed are as shown in table 3, show separation
Antioxidation polypeptide molecular weight distribution is between 330.0-3388.3, wherein (intensity value exists between being mainly distributed on 342.4-456.7
More than 1000), i.e., between serial number the 2nd to 10, it is shown to be the small peptide of low molecular weight.
Mass-to-charge ratio, signal-to-noise ratio and the intensity of peptide molecule after table 3 detaches
Embodiment 4
(1) after wheat embryo being carried out micro-wave vacuum, ultramicro grinding, wheat embryo Ultramicro-powder is obtained, took 60 mesh
The sample 100g of sieve, using 1.2mol/L sodium chloride, pH value 11, solid-liquid ratio 1：15, sample is carried using ultrasonic wave added method
30min is taken, ultrasonic wave uses 5kW, repeats extraction 2 times, obtains wheat plantule protein solution；
(2) separation of solid and liquid is carried out using 10000rpm centrifugations 60min, takes supernatant, as wheat plantule protein extracting solution；
(3) the wheat plantule protein extracting solution 2800mL that step (2) obtains is taken to be concentrated by ultrafiltration, 40 DEG C of temperature, operation
Pressure is 0.7MPa, and ultrafiltration membrane aperture is 200nm, is concentrated into 900mL, obtains wheat plantule protein concentrate；
(4) pepsin of concentrate mass ratio 0.25% is accounted for the addition of wheat plantule protein concentrate, adjusts pH value
1.0,60 DEG C of heat preservations digest 120min, and the antioxidant activity of supernatant, and 80 DEG C of heat preservation 30min enzyme deactivations are measured after enzymolysis, are obtained
Obtain germ protein peptide solution；
(5) solution that step (4) obtains further is concentrated by ultrafiltration, operating pressure 0.7MPa, ultrafiltration membrane aperture is
200nm obtains the solution 500mL containing polypeptide, is chromatographed using DEAE-Sepharose F.F ion-exchangers, with 0.1-
0.5mol/L sodium chloride is eluted, and the free radical scavenging activity of different elution fractions (number 1-5) is as shown in table 4, is detached
The polypeptide of purifying, wherein the polypeptide Scavenging ability with 2-5 portion collections is stronger.The part of polypeptide is carried out infrared true
Vacuum freecing-dry obtains antioxidation polypeptide sample.Sample measures its 4th sample segment molecular weight distribution by MALDI-TOF-MS
Figure is as shown in Figure 6.From fig. 6 it can be seen that molecular weight distribution is mainly between 363.5-661.8, wherein again with 448.8 Hes
Based on 490.0, relative intensity highest, the small peptide molecular weight for acquisition.
The different DPPH free radical scavenging activities for collecting part of table 4
| Number | DPPH free radical scavenging activities | Hydroxyl radical free radical Scavenging activity |
| 1 | 9.84 | 3.8 |
| 2 | 19.60 | 10.5 |
| 3 | 22.42 | 11.2 |
| 4 | 28.27 | 13.8 |
| 5 | 35.72 | 17.4 |
Claims (9)
1. a kind of preparation method of wheat embryo anti-oxidation peptide, which is characterized in that this method includes the following steps：
(1) after wheat embryo being carried out micro-wave vacuum, ultramicro grinding, wheat embryo Ultramicro-powder is obtained；Using basic salt method,
Wheat germ powder is extracted with reference to ultrasonic wave added method, obtains wheat plantule protein solution；
(2) separation of solid and liquid takes supernatant, as wheat plantule protein extracting solution；
(3) the wheat plantule protein extracting solution obtained to step (2) is concentrated by ultrafiltration, and obtains wheat plantule protein concentrate；
(4) pepsin is added in above-mentioned concentrate, carries out the enzymolysis of albumen, it is molten to obtain germ protein peptide for enzyme deactivation after enzymolysis
Liquid；
(5) wheat plantule protein peptide solution is concentrated by ultrafiltration, chromatographs filtering and drying, the wheat embryo antioxygen purified
Change peptide.
2. the preparation method of wheat embryo anti-oxidation peptide according to claim 1, which is characterized in that the step (1)
The leaching process is specially：Alkaline salt solution and the liquid material volume ratio of wheat germ powder are used as 5~15：1, water-bath adds
Heat, extraction time 30-120min, extracting times 2~3 times.
3. the preparation method of wheat embryo anti-oxidation peptide according to claim 1 or 2, which is characterized in that the step
(1) pH value range of alkaline salt solution is 8-11, salinity 0.2-1.2mol/L used by neutral and alkali salt method.
4. the preparation method of wheat embryo anti-oxidation peptide according to any one of claim 1-3, which is characterized in that described
The step of (1) in ultrasonic power be 30W-5KW, ultrasonic time 30-120min.
5. the preparation method of wheat embryo anti-oxidation peptide according to claim 1, which is characterized in that the step (2)
Separation of solid and liquid is carried out using the method for centrifugation or plate compression.
6. the preparation method of wheat embryo anti-oxidation peptide according to claim 1, which is characterized in that the step (3),
(5) operating pressure of ultrafiltration concentration is 0.1-0.7MPa, and ultrafiltration membrane aperture is 10-200nm.
7. the preparation method of wheat embryo anti-oxidation peptide according to claim 1, which is characterized in that the step (4)
Middle hydrolysis temperature is 30-60 DEG C, enzymolysis time 1-6h, and enzyme adding proportion is the 0.1-1% of step (3) concentrate.
8. the preparation method of wheat embryo anti-oxidation peptide according to claim 1, which is characterized in that the step (4)
In enzyme deactivation condition for 80-100 DEG C heat preservation 5-20min.
9. the preparation method of wheat embryo anti-oxidation peptide according to claim 1, which is characterized in that the step (5)
In chromatography filtering, the combination filtered, or both using gel filtration or ion exchange chromatography.
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| CN110331181A (en) * | 2019-08-12 | 2019-10-15 | 河南工业大学 | A kind of preparation method of purple spring wheat wheat bran anti-oxidation peptide |
| CN111096390A (en) * | 2019-12-27 | 2020-05-05 | 广元市剑粮面业有限公司 | Extraction method of wheat germ peptide |
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| CN112825997A (en) * | 2021-01-08 | 2021-05-25 | 古浪伊禧堂伟业生物科技有限公司 | Wheat germ compound peptide solid beverage and preparation method thereof |
| US20230008335A1 (en) * | 2021-07-08 | 2023-01-12 | Nanjing University Of Finance And Economics | Oligopeptide with anti-inflammatory activity, preparation method, and application thereof |
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| CN109198038A (en) * | 2018-11-02 | 2019-01-15 | 安徽鑫乐源食品有限公司 | A kind of preparation method roasting fragrant plumule soya-bean milk |
| CN110331181A (en) * | 2019-08-12 | 2019-10-15 | 河南工业大学 | A kind of preparation method of purple spring wheat wheat bran anti-oxidation peptide |
| CN111096390A (en) * | 2019-12-27 | 2020-05-05 | 广元市剑粮面业有限公司 | Extraction method of wheat germ peptide |
| CN111317057A (en) * | 2020-03-20 | 2020-06-23 | 江南大学 | Method for extracting rice protein from brown rice |
| CN111317057B (en) * | 2020-03-20 | 2022-12-27 | 江南大学 | Method for extracting rice protein from brown rice |
| CN112825997A (en) * | 2021-01-08 | 2021-05-25 | 古浪伊禧堂伟业生物科技有限公司 | Wheat germ compound peptide solid beverage and preparation method thereof |
| US20230008335A1 (en) * | 2021-07-08 | 2023-01-12 | Nanjing University Of Finance And Economics | Oligopeptide with anti-inflammatory activity, preparation method, and application thereof |
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